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Human Phospho-MAPK Array Kit
Proteome ProfilerTM Array
This package insert must be read in its entirety before using this product. For research use only. Not for use in diagnostic procedures.
Catalog Number ARY002B
For the parallel determination of the relative levels of phosphorylation of Mitogen-Activated Protein Kinases (MAPKs) and other serine/threonine kinases.
MANUFACTURED AND DISTRIBUTED BY:
USA & Canada | R&D Systems, Inc. 614 McKinley Place NE, Minneapolis, MN 55413, USATEL: (800) 343-7475 (612) 379-2956 FAX: (612) 656-4400E-MAIL: [email protected]
DISTRIBUTED BY:
UK & Europe | R&D Systems Europe, Ltd.19 Barton Lane, Abingdon Science Park, Abingdon OX14 3NB, UKTEL: +44 (0)1235 529449 FAX: +44 (0)1235 533420E-MAIL: [email protected]
China | R&D Systems China Co., Ltd.24A1 Hua Min Empire Plaza, 726 West Yan An Road, Shanghai PRC 200050TEL: +86 (21) 52380373 FAX: +86 (21) 52371001E-MAIL: [email protected]
TABLE OF CONTENTS
SECTION PAGE
INTRODUCTION .....................................................................................................................................................................1PRINCIPLE OF THE ASSAY ...................................................................................................................................................1TECHNICAL HINTS .................................................................................................................................................................1MATERIALS PROVIDED & STORAGE CONDITIONS ...................................................................................................2OTHER SUPPLIES REQUIRED .............................................................................................................................................2PRECAUTIONS .........................................................................................................................................................................3SAMPLE COLLECTION & STORAGE .................................................................................................................................3REAGENT PREPARATION .....................................................................................................................................................3ARRAY PROCEDURE .............................................................................................................................................................4DATA ANALYSIS ......................................................................................................................................................................6PROFILING PROTEIN PHOSPHORYLATION IN CELL LYSATES ...............................................................................7PATHWAY INHIBITION ..........................................................................................................................................................9APPENDIX .............................................................................................................................................................................. 10
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INTRODUCTIONAnalyzing the phosphorylation status of all three major families of mitogen-activated protein kinases (MAPKs), the extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNK1-3), and different p38 isoforms (α/β/δ/γ), is essential in understanding the roles these signaling molecules play in mechanisms underlying cell function and disease. Other intracellular proteins, such as Akt, GSK-3, p70 S6 Kinase, TOR, p53, and CREB, are additional important regulators of signal transduction, mediating development and cell proliferation. The Human Phospho-MAPK Array is a rapid, sensitive, and economical tool used to simultaneously detect the relative levels of phosphorylation of 26 kinases including nine MAPKs without performing numerous immunoprecipitations or Western blots. Each capture antibody was carefully selected using lysate samples prepared from cell lines known to express the target proteins.
PRINCIPLE OF THE ASSAYCapture and control antibodies have been spotted in duplicate on nitrocellulose membranes. Cell lysates are diluted, mixed with a cocktail of biotinylated detection antibodies, and incubated overnight with the Proteome Profiler Human Phospho-MAPK Array. The membrane is washed to remove unbound material. Streptavidin-HRP and chemiluminescent detection reagents are applied, and a signal is produced at each capture spot corresponding to the amount of phosphorylated protein bound. Refer to the Appendix for a list and coordinates of analytes and controls.
TECHNICAL HINTS• FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.• This kit should not be used beyond the expiration date on the kit label.• Do not mix or substitute reagents with those from other lots or sources. Substitution of
some high intensity chemiluminescent reagents for Chemi Reagents 1 and 2 may cause either increased background or diminished signal depending on the reagent.
• Any variation in sample handling, buffers, operator, pipetting technique, washing technique, and incubation time or temperature can alter the performance of the kit.
• The Human Phospho-MAPK Array membranes are validated for single use only.• Always use gloved hands and flat-tipped tweezers to handle the membranes.• Pick up the membranes from the edge on the side with the identification number avoiding
the area with the printed antibodies.• A thorough and consistent wash technique is essential for proper assay performance.
Individual membranes should be washed in separate containers to minimize background. Wash Buffer should be removed completely from the membrane before proceeding to the next step.
• Do not allow the membrane to dry out. This will cause high background.• Avoid microbial contamination of reagents and buffers.• For a procedure demonstration video, please visit:
www.RnDSystems.com/ProteomeProfilerVideo.
For research use only. Not for use in diagnostic procedures.2
MATERIALS PROVIDED & STORAGE CONDITIONSStore the unopened kit at 2-8 °C. Do not use past kit expiration date.
PART PART # AMOUNT PROVIDEDSTORAGE OF OPENED/ RECONSTITUTED MATERIAL
Human Phospho-MAPK Array 893909 4 nitrocellulose membranes each containing 26 different capture antibodies printed in duplicate.
Return unused membranes to the foil pouch containing the desiccant pack. Reseal along entire edge of the zip-seal. May be stored for up to 3 months at 2-8 °C.*
Array Buffer 1 895477 21 mL of a buffered protein base with preservatives.
May be stored for up to 3 months at 2-8 °C.*
Array Buffer 5 895876 21 mL of a buffered protein base with preservatives.
Lysis Buffer 6 895561 21 mL of a denaturing buffered solution.
Wash Buffer Concentrate 895003 2 vials (21 mL/vial) of a 25-fold concentrated solution of buffered surfactant with preservative. May turn yellow over time.
Detection Antibody Cocktail, Human Phospho-MAPK Array
893910 1 vial of a biotinylated antibody cocktail; lyophilized.
Streptavidin-HRP 893019 200 μL of streptavidin conjugated to horseradish-peroxidase.
Chemi Reagent 1 894287 2.5 mL of stabilized hydrogen peroxide with preservative.
Chemi Reagent 2 894288 2.5 mL of stabilized luminol with preservative.
4-Well Rectangular Multi-dish 607544 Clear 4-well rectangular multi-dish.Store at room temperature.Transparency Overlay Template 607725 1 transparency overlay template for
coordinate reference.* Provided this is within the expiration date of the kit.
OTHER SUPPLIES REQUIRED• Pipettes and pipette tips• Gloves• Phosphate-Buffered Saline (PBS)• Deionized or distilled water• Flat-tipped tweezers• Rocking platform shaker• Microcentrifuge• Plastic containers with the capacity to hold
50 mL (for washing the arrays)• Plastic transparent sheet protector (trimmed
to 10 cm x 12 cm and open on three sides)• Plastic wrap
• Absorbent lab wipes (KimWipes® or equivalent)
• Paper towels• Autoradiography cassette• Film developer• X-ray film (Kodak® BioMax™ Light-1,
Catalog # 1788207) or equivalent• Flatbed scanner with transparency adapter
capable of transmission mode• Computer capable of running image analysis
software and Microsoft® Excel
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PRECAUTIONSChemi Reagents 1 and 2 contain Boric Acid which is suspected of damaging fertility or the unborn child.
Some components in this kit contain ProClin® which may cause an allergic skin reaction. Avoid breathing mist.
Wear protective gloves, clothing, eye, and face protection. Wash hands thoroughly after handling. Please refer to the MSDS on our website prior to use.
SAMPLE COLLECTION & STORAGEThe sample collection and storage conditions listed below are intended as general guidelines. Sample stability has not been evaluated.
Since the Human Phospho-MAPK Array detects relative phosphorylation levels of individual analytes, it is important to include appropriate control samples.
Note: Sample amount may be empirically adjusted to attain optimal sensitivity with minimal background. The suggested starting range for cell lysates is 100-300 μg.
Cell Lysates - Rinse cells with PBS, making sure to remove any remaining PBS before adding lysis buffer. Solubilize the cells at 1 x 107 cells/mL in Lysis Buffer 6. Pipette up and down to resuspend and rock the lysates gently at 2-8 °C for 30 minutes. Microcentrifuge at 14,000 x g for 5 minutes, and transfer the supernate into a clean test tube. Quantitation of sample protein concentrations using a total protein assay is recommended. Assay immediately or aliquot and store at ≤ -70 °C. Avoid repeated freeze-thaw cycles.
REAGENT PREPARATIONBring all reagents to room temperature before use.
Human Phospho-MAPK Array - Four nitrocellulose membranes each containing 26 different capture antibodies printed in duplicate. Handle the membranes only with gloved hands and flat-tipped tweezers.
Detection Antibody Cocktail - One vial of lyophilized biotinylated phospho-specific antibodies. Before use, reconstitute the Detection Antibody Cocktail in 100 μL of deionized or distilled water.
1X Wash Buffer - If crystals have formed in the concentrate, warm the bottles to room temperature and mix gently until the crystals have completey dissolved. Dilute 40 mL of 25X Wash Buffer Concentrate into 960 mL of deionized or distilled water.
Chemi Reagent Mix - Chemi Reagent 1 and 2 should be mixed in equal volumes within 15 minutes of use. Protect from light. 1 mL of the resultant mixture is required for each membrane.
For research use only. Not for use in diagnostic procedures.4
ARRAY PROCEDURE Bring all reagents to room temperature before use. Keep samples on ice. To avoid contamination, wear gloves while performing the procedures.
1. Prepare all reagents and samples as directed in the previous sections.
2. Pipette 2.0 mL of Array Buffer 5 into each well of the 4-Well Multi-dish to be used. Array Buffer 5 serves as a block buffer.
3. Using flat-tip tweezers, remove each membrane to be used from between the protective sheets and place in a well of the 4-Well Multi-dish. The number on the membrane should be facing upward.
Note: Upon contact with Array Buffer 5, the blue dye from the spots will disappear, but the capture antibodies are retained in their specific locations.
4. Incubate for one hour on a rocking platform shaker. Orient the tray so that each membrane rocks end to end in its well.
5. While the membranes are blocking, prepare samples by adding up to 400 μL of each sample to separate tubes. Adjust to a final volume of 1.5 mL with Array Buffer 1.
6. Add 20 μL of reconstituted Detection Antibody Cocktail to each prepared sample. Mix and incubate at room temperature for one hour.
7. Aspirate Array Buffer 5 from the wells of the 4-Well Multi-dish and add the prepared sample/antibody mixtures. Place the lid on the 4-Well Multi-dish.
8. Incubate overnight at 2-8 °C on a rocking platform shaker.
Note: A shorter incubation time may be used if optimal sensitivity is not required.
9. Carefully remove each membrane and place into individual plastic containers with 20 mL of 1X Wash Buffer. Rinse the 4-Well Multi-dish with deionized or distilled water and dry thoroughly.
10. Wash each membrane with 1X Wash Buffer for 10 minutes on a rocking platform shaker. Repeat two times for a total of three washes.
11. Dilute the Streptavidin-HRP in Array Buffer 5 using the dilution factor on the vial label. Pipette 2.0 mL of diluted Streptavidin-HRP into each well of the 4-Well Multi-dish.
12. Carefully remove each membrane from its wash container. Allow excess Wash Buffer to drain from the membrane. Return the membrane to the 4-Well Multi-dish containing the diluted Streptavidin-HRP. Cover the wells with the lid.
13. Incubate for 30 minutes at room temperature on a rocking platform shaker.
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ARRAY PROCEDURE CONTINUED14. Wash each array as described in steps 9 and 10.
Note: Complete the remaining steps without interruption.
15. Carefully remove each membrane from its wash container. Allow excess Wash Buffer to drain from the membrane by blotting the lower edge onto paper towels. Place each membrane on the bottom sheet of the plastic sheet protector with the identification number facing up.
16. Pipette 1 mL of the prepared Chemi Reagent Mix evenly onto each membrane.
Note: Using less than 1 mL of Chemi Reagent Mix per membrane may result in incomplete membrane coverage.
17. Carefully cover with the top sheet of the plastic sheet protector. Gently smooth out any air bubbles and ensure Chemi Reagent Mix is spread evenly to all corners of each membrane. Incubate for 1 minute.
18. Position paper towels on top and sides of plastic sheet protector containing the membranes and carefully squeeze out excess Chemi Reagent Mix.
19. Remove the top plastic sheet protector and carefully lay an absorbent lab wipe on top of the membranes to blot off any remaining Chemi Reagent Mix.
20. Leaving membranes on the bottom plastic sheet protector, cover the membranes with plastic wrap taking care to gently smooth out any air bubbles. Wrap the excess plastic wrap around the back of the sheet protector so that the membranes and sheet protector are completely wrapped.
21. Place the membranes with the identification numbers facing up in an autoradiography film cassette.
Note: Use an autoradiography cassette that is not used with radioactive isotope detection.
22. Expose membranes to X-ray film for 1-10 minutes. Multiple exposure times are recommended.
For research use only. Not for use in diagnostic procedures.6
DATA ANALYSISThe positive signals seen on developed film can be quickly identified by placing the transparency overlay on the array image and aligning it with the pairs of reference spots in three corners of each array. The stamped identification number on the membrane should be placed on the left hand side. The location of controls and capture antibodies is listed in the Appendix.
Note: Reference spots are included to align the transparency overlay template and to demonstrate that the array has been incubated with Streptavidin-HRP during the assay procedure.
Pixel densities on developed X-ray film can be collected and analyzed using a transmission-mode scanner and image analysis software.
1. Create a template to analyze pixel density in each spot of the array.
2. Export signal values to a spreadsheet file for manipulation in a program such as Microsoft Excel.
3. Determine the average signal (pixel density) of the pair of duplicate spots representing each kinase.
4. Subtract an averaged background signal from each spot. Use a signal from a clear area of the array or negative control spots as a background value.
5. Compare corresponding signals on different arrays to determine the relative change in protein phosphorylation between samples.
ABCDE
F
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Human Phospho-MAPK Array Coordinates
This image is not to scale. It is for coordinate reference only. Please use the transparency overlay for analyte identification.
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PROFILING PROTEIN PHOSPHORYLATION IN CELL LYSATESThe Human Phospho-MAPK Array detects multiple phosphorylated proteins in untreated and treated cell lysates. All arrays were incubated with 200 μg of lysate.
CREB
ERK1
ERK2
HSP2
7
JNK1
JNK2
JNK p
an
MKK6
MSK2
p38α
p38γ
TOR
Pixe
l Den
sity
0
5000
10,000
15,000
20,000
25,000
30,000
35,000UntreatedUV Treated
HeLa Cells (Untreated)
HeLa Cells (UV Treated)
1
2
3
4
1 2 3
45
5
6
6 7
7
8
8 9
10
9
10
12
11 12
HeLa Cells (Untreated vs. UV Treated)
A
11
Figure 1A: HeLa human cervical epithelial carcinoma cells were either untreated or exposed to 150 J/m2 of UV light followed by a 30 minute recovery period before lysis. Data shown are from a 2 minute exposure to X-ray film.
CREB
ERK1
ERK2
GSK-
3α/β
GSK-
3β
JNK2
JNK p
an
p38α
p70 S
6 Kina
se
TOR
Pixe
l Den
sity
0
5000
10,000
15,000
20,000
25,000
30,000
35,000UntreatedUV TreatedMCF-7 Cells (Untreated)
MCF-7 Cells (UV Treated)
1 2
3
4
1 2 3 4 5
5
6
6
7
8
7
9
9
10
MCF-7 Cells (Untreated vs. UV Treated)
10
B
8
Figure 1B: MCF-7 human breast cancer cells were either untreated or exposed to 50 J/m2 of UV light followed by a 4 hour recovery period before lysis. Data shown are from a 1 minute exposure to X-ray film.
For research use only. Not for use in diagnostic procedures.8
JNK1
JNK2
JNK p
an
MSK2
p38α
p38δ
p38γ
p53 (
S46)
Pixe
l Den
sity
0
5000
10,000
15,000
20,000
25,000
30,000UntreatedrhIL-1β Treated
HepG2 Cells (Untreated)
HepG2 Cells (rhIL-1β Treated) 1 2
3
41
2 34
5
5
6 78
86 7
CHepG2 Cells Untreated and Treated with 25 ng/mL rhIL-1β (30 minutes)
Figure 1C: HepG2 human hepatocellular carcinoma cells were either untreated or treated with 25 ng/mL of recombinant human (rh) IL-1β (R&D Systems, Catalog # 201-LB) for 30 minutes. Data shown are from a 2 minute exposure to X-ray film.
PROFILING PROTEIN PHOSPHORYLATION IN CELL LYSATES CONTINUED
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HeLa Cells Untreated and PMA Treatedwith and without MEK Inhibitor U0126
CREB
ERK1
ERK2
HSP2
7
MSK2
p38α
RSK1
TOR
Pixe
l Den
sity
0
10,000
20,000
30,000
40,000UntreatedPMA TreatedPMA + U0126 Treated
HeLa Cells (Untreated)
HeLa Cells (PMA Treated; 200 nM)
1
23
4
1 2 34
5
5 6
6 7
7
88
HeLa Cells (PMA Treated; 200 nM+75 μM U0126)
4
6 7 8
5
A
1 2 3
Figure 2A: HeLa human cervical epithelial carcinoma cells were either untreated or treated with 200 nM PMA for 20 minutes either with or without the MEK inhibitor U0126. Data shown are from a 5 minute exposure to X-ray film.
PATHWAY INHIBITIONThe Human Phospho-MAPK Array shows the effect of inhibitors on specific pathways. All arrays were incubated with 200 μg of lysate.
MCF-7 Cells Untreated and rhIGF-I Treatedwith and without PI3K Inhibitor LY294002
Akt1
Akt2
Akt p
an
ERK1
ERK2
GSK-
3α/β
GSK-
3β
p70 S
6 Kina
se TOR
Pixe
l Den
sity
0
10,000
20,000
30,000
40,000
50,000UntreatedrhIGF-I TreatedrhIGF-I Treated withLY294002 Pre-treatment
MCF-7 Cells (Untreated)
MCF-7 Cells (rhIGF-I Treated; No Pre-treatment)
1
2
3
4
1 2 3 4 5
5
6
6 7
7
8
8 9
9MCF-7 Cells (rhIGF-I Treated; Pre-treated with LY294002)
B
1 2 3 4 5 6 7
8 9
Figure 2B: MCF-7 human breast cancer cells were either untreated or treated with 100 ng/mL of rhIGF-I (R&D Systems, Catalog # 291-G1) for 1 hour. Cells for rhIGF-I treatment either received a 1 hour pre-treatment with the PI3K inhibitor, LY294002, or received no pre-treatment. Data shown are from a 6 minute exposure to X-ray film.
For research use only. Not for use in diagnostic procedures.10
APPENDIXRefer to the table below for the Human Phospho-MAPK Array coordinates.
Coordinate Target/Control Alternate Nomenclature Phosphorylation Site Detected
A1, A2 Reference Spots ___ ___
A21, A22 Reference Spots ___ ___
B3, B4 Akt1 PKBα, RACα S473B5, B6 Akt2 PKBβ, RACβ S474B7, B8 Akt3 PKBγ, RACγ S472B9, B10 Akt pan ___ S473, S474, S472B11, B12 CREB ___ S133B13, B14 ERK1 MAPK3, p44 MAPK T202/Y204B15, B16 ERK2 MAPK1, p42 MAPK T185/Y187B17, B18 GSK-3α/β GSK3A/GSK3B S21/S9B19, B20 GSK-3β GSK3B S9C3, C4 HSP27 HSPB1, SRP27 S78/S82C5, C6 JNK1 MAPK8, SAPK1γ T183/Y185C7, C8 JNK2 MAPK9, SAPK1α T183/Y185C9, C10 JNK3 MAPK10, SAPK1β T221/Y223C11, C12 JNK pan ___ T183/Y185, T221/Y223C13, C14 MKK3 MEK3, MAP2K3 S218/T222C15, C16 MKK6 MEK6, MAP2K6 S207/T211C17, C18 MSK2 RSKβ, RPS6KA4 S360D3, D4 p38α MAPK14, SAPK2A, CSBP1 T180/Y182D5, D6 p38β MAPK11, SAPK2B, p38-2 T180/Y182D7, D8 p38δ MAPK13, SAPK4 T180/Y182D9, D10 p38γ MAPK12, SAPK3, ERK6 T183/Y185D11, D12 p53 ___ S46D13, D14 p70 S6 Kinase S6K1, p70α, RPS6KB1 T421/S424D15, D16 RSK1 MAPKAPK1α, RPS6KA1 S380D17, D18 RSK2 ISPK-1, RPS6KA3 S386D19, D20 TOR ___ S2448E19, E20 PBS Control (-) ___
F1, F2 Reference Spots ___ ___
10.10 752140.4 3/14
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